Estrogen sulfotransferase (EST) catalyzes the sulfation of estrogens such as estradiol and estrone, using the cofactor 3'-phosphoandenosine- 5'-phosphosulfate (PAPS). Since sulfated hormones and chemicals are generally less active and toxic, sulfation is considered to contribute to metabolic inactivation and ultimately to excretion. Recent reports show, however, that steroid sulfation may play positive roles in biological processes beyond merely inactivation and excretion. For example, while estrone-3-sulfate may elicit a unique biological effect in uterine endometrium, pregnenolone sulfate (a neurosteroid) can suppress the GABA receptor by binding to it. As a result, steroid sulfotransferases are involved in maintaining endocrine homeostasis as well as in providing biologically active signals. Additionally, as a result of the catabolic role of EST, environmental agents which act as inhibitors of this enzyme may be estrogenic. A system has been developed for expressing high levels of mammalian recombinant EST in E. coli. Initial 1H NMR analyses of the EST, a monomeric enzyme of MW ~ 35,000, show five resonances in the region expected for the histidine H-2 protons. Further, it became apparent that the preparations contain significant amounts of the PAPS cofactor (or related structures) which are bound sufficiently well so that they are present in purified enzyme samples. There is also some evidence for the presence of other small ligands. The initial series of studies will be directed toward characterizing the interactions of EST with PAPS and with various estrogen analogs, and with the assignment of the histidine resonances using site-directed mutagenesis. Longer term progress in structural elucidation requires further improvement in the level of protein expression.